Intravenous injection (i.v.) of soluble proteins can induce antigen-specific tolerance in experimental autoimmune encephalomyelitis (EAE). I.V. tolerance effectively prevents or suppresses the disease. Dendritic cells (DCs) play a crucial role in initiation and regulation of adaptive immune responses. There are two distinct subsets of DCs in murine spleens: CD8+CD11c+CD11b- and CD8-CD11c+CD11b+. We have shown that induction of i.v. tolerance expands subtype of CD11c+CD11b+ DCs in the spleen and CNS of mice immunized for EAE. Adoptive transfer of these cells into mice with ongoing EAE rapidly ameliorates the disease and induces strong Treg and Th2 responses. These results demonstrate that CD11c+CD11b+ cells are a potent tolerogenic DC subset, and warrant further research on their mechanisms of action. The central hypothesis of this proposal is that CD11c+CD11b+ DCs play a major role in i.v. tolerance in EAE. To test this hypothesis, the following specific aims are proposed: Aim 1. To determine the role of apoptotic T cells in the induction of tolerogenic CD11c+CD11b+ DCs in EAE. We have shown that i.v. injection of myelin antigen into mice with EAE induced by the same antigen results in: a) apoptosis of a significant number of activated myelin-specific T cells;and b) generation of CD11c+CD11b+ DCs. We hypothesize that DCs which phagocytize apoptotic T cells acquire tolerogenic CD11c+CD11b+ phenotype. Aim 2. To study the role of CD11c+CD11b+ DCs in antigen specificity of i.v. tolerance. I.V. tolerance is antigen specific. T cells play an important role in this process;however, we have shown that adoptively transferred CD11c+CD11b+ DCs from tolerized mice suppress EAE. We hypothesize that tolerogenic DCs convert Th1 and Th17 cells to Th2 and Treg phenotype in an antigen-specific manner. Aim 3. To study the role of soluble mediators in tolerogenic action of CD11c+CD11b+ DCs. It is not clear whether it is cell-cell contact between tolerogenic CD11c+CD11b+ DCs and antigen-specific T cells or soluble mediators produced by DCs that mediate tolerance. We hypothesize that tolerogenic DCs act both by cell-cell contact and by secretion of soluble factors to induce tolerance. These studies should result in a better understanding of the role of dendritic cells in i.v. tolerance, and provide an insight into their potential use as a therapy for human autoimmune diseases. PUBLIC HEALTH RELEVANCE Intravenous injection (i.v.) of soluble proteins can induce antigen-specific tolerance, a phenomenon termed "i.v. tolerance." In experimental autoimmune encephalomyelitis (EAE), which is an animal model of human multiple sclerosis, i.v. tolerance effectively prevents disease. The exact mechanisms underlying i.v. tolerance suppression of disease are not well understood. Dendritic cells (DCs) are immune cells that play a crucial role in regulation of immune responses. We showed that induction of i.v. tolerance induces a particular type of DC and that transfer of these DCs into mice with ongoing EAE rapidly suppresses disease. Our results suggest that these DCs play a crucial role in i.v. tolerance. We propose to investigate mechanisms underlying generation of DCs in i.v. tolerance, and in which way(s) they suppress disease. These studies should result in a better understanding of the role of dendritic cells in i.v. tolerance, and provide an insight into their potential use as a therapy for human autoimmune diseases.